Eukaryotic and prokaryotic cells alike possess the remarkable ability to alter their phenotypes through global changes in gene expression. The proper execution of transitions in gene expression is critical for cellular transformation, eithe as part of normal cell and developmental biology, or, abnormally, as during the conversion of a normal cell to a cancer cell. In bacteria, transitions in gene expression programs drive phenotypic changes critical for growth, development, and pathogenesis. Despite their importance, there are few examples of transitions in gene expression that are understood in molecular detail from beginning to end. In order to close this critical gap in knowledge, we will elucidate the molecular mechanisms that control the switch from early (F-directed) to late (G-directed) transcription in the developing Bacillus subtilis spore. Broadly speaking, the transition to a new gene expression program requires not only that the new program is induced, but also that the old gene expression program is deactivated. While significant effort has been placed on identifying the mechanisms that activate G, very little is known of the mechanisms by which F is deactivated and how these two events are coordinated. The central hypothesis of our study is that proper transition from F to G requires regulatory mechanisms to first prevent G activity at early times and subsequently to disarm the F protein at or before the switch to late gene expression. We will test this central hypothesis using a combination of genetic, biochemical, and cell-biological approaches. In Specific Aim 1, we will determine the mechanism by which G activity is restricted at early times. The working hypothesis for this aim is that negative transcriptional or translational regulation prevents significant accumulation of G protein prior t the switch to late gene expression. In Specific Aim 2, we will identify the mechanism by which F in - a small, conserved protein required for the switch from F to G - disarms . Finally, in Specific Aim 3, we will distinguish two competing models for a second, G -dependent but Fin- independent, mechanism of F inhibition: that G outcompetes F for access to RNA polymerase or, alter- natively, that G mediates F inhibition via activation of one or more target genes. In all, the expected out- come of the proposed research is significant mechanistic insight into how the switch from F - to G-directed gene expression is achieved in the developing B. subtilis spore. This contribution will be significant because it will address a fundamental, yet currently inadequately answered question in cell and developmental biology: How, at the molecular level, do cells shed their old identity while simultaneously taking on a new one? Moreover, this study has the potential to identify novel antibiotic targets for gram-positive spore forming bacterial pathogens such as Bacillus anthracis and Clostridium difficile. Finally, this research plan is innovative not only because it addresses largely overlooked aspects of gene expression switches, but also because it will be carried out exclusively by graduate and undergraduate students. PUBLIC HEALTH RELEVANCE: Properly regulated transitions in gene expression are critical for normal cellular growth and development, as well as for bacterial pathogenesis. Here we will elucidate the molecular mechanisms that control the switch from early to late developmental transcription during spore formation by the bacterium Bacillus subtilis, a premier model system for studies of regulation. This project is relevant to NIH's mission because it will address a fundamental, yet currently inadequately understood aspect of cell and developmental biology, and, furthermore, has significant potential to identify novel antibiotic targets in gram-positive spore-forming bacterial pathogens such as Bacillus anthracis and Clostridium difficile.